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Peroxisome Proliferator Activator Receptor- Agonists and 15-Deoxy-^12,1412,14-PGJ₂ Induce Apoptosis in Normal and Malignant B-Lineage Cells¹

Josué Padilla^*,,¶, Kuljeet Kaur^*, H. James Cao^||, Terry J. Smith^|| and Richard P. Phipps^2,*,,,§

^* University of Rochester Cancer Center and Departments of Microbiology and Immunology, Pediatrics, ^§ Environmental Medicine, and ^¶ Periodontology, Eastman Department of Dentistry, Rochester, NY 14642; and ^|| Division of Molecular Medicine, University of California, Los Angeles School of Medicine, Harbor-University of California Los Angeles Medical Center, Torrance, CA 90502

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor- (PPAR-) on B-lineage cells. Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation (e.g., 70Z/3, CH31, WEHI-231, CH12, and J558) express PPAR- mRNA and, by Western blot analysis, the 67-kDa PPAR- protein. 15-Deoxy-^12,14-PGJ₂ (15d-PGJ₂), a PPAR- agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [³H]thymidine and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. Only PPAR- agonists (thiazolidinediones), and not PPAR- agonists, mimicked the effect of 15d-PGJ₂ on B-lineage cells, indicating that the mechanism by which 15d-PGJ₂ negatively affects B-lineage cells involves in part PPAR-. The mechanism by which PPAR- agonists induce cytotoxicity is via apoptosis, as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. Interestingly, addition of PGF₂, which was not known to affect lymphocytes, dramatically attenuated the deleterious effects of PPAR- agonists on B lymphomas. Surprisingly, 15d-PGJ₂ induced a massive increase in nuclear mitogen-activated protein kinase activation, and pretreatment with PGF₂ blunted the mitogen-activated protein kinase activation. This is the first study evaluating PPAR- expression and its significance on B lymphocytes. PPAR- agonists may serve as a counterbalance to the stimulating effects of other PGs, namely PGE₂, which promotes B cell differentiation. Finally, the use of PGs, such as 15d-PGJ₂, and synthetic PPAR- agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prostaglandin D₂ is the major metabolite of arachidonic acid secreted by mast cells (1) and is a major product of macrophages (2). PGD₂ is the major PG synthesized by bone marrow (3, 4). It readily undergoes dehydration in vivo and in vitro to additional, biologically active PGs of the J₂ series (5). Metabolites of the J₂ series have their own unique spectrum of biological activities, including inhibition of cell cycle progression, suppression of viral replication, induction of heat shock proteins, and stimulation of osteogenesis (5). While PGD₂ as well as other PGs signal cells through G protein-coupled receptors (6), the PGJ₂ series is actively incorporated into the nucleus and associates with nuclear proteins by unknown mechanisms (7). 15-Deoxy-^12,14-PGJ₂ (15d-PGJ₂)³ is the final metabolite of PGD₂ degradation. Both 15d-PGJ₂ and the antidiabetic drugs of the thiazolidinedione class are potent ligands for a nuclear hormone receptor known as peroxisome proliferator-activated receptor (PPAR-). PPAR- is a member of a superfamily of nuclear hormone receptors that function as ligand-dependent transcription factors (8). Three receptor subtypes of PPARs, termed , , and , have been identified. The transcriptional activity of the PPAR subtypes is enhanced by a multitude of chemical compounds, including fatty acids, thiazolidinedione antidiabetic agents, selected PGs, peroxisome proliferators, and fibrate hypolipidemic drugs (8). After activation, PPARs control the expression of genes implicated in extra- and intracellular lipid metabolism. These genes encode enzymes involved in the peroxisomal ß-oxidation pathway (8). In particular, PPAR- is abundantly expressed in adipocytes and functions as a key regulator of adipocyte differentiation (9). 15d-PGJ₂ and thiazolidinediones, such as troglitazone and ciglitazone, bind directly to PPAR and promote adipogenesis of cultured fibroblasts (9). Recently, the expression of PPAR- has been detected on macrophages, T cells, endothelial cells, vascular smooth muscle cells, and colonic tumor cells (10, 11, 12, 13, 14, 15, 16). The significance and expression of PPAR- in the immune-related cells is poorly understood. Moreover, there are no published data on PPAR expression by B-lineage cells. In this study 15d-PGJ₂ as well as the synthetic thiazolidinediones are shown to be cytotoxic for B cells and to induce cell death in an apoptotic fashion. The mechanism by which 15d-PGJ₂ interacts with B-lineage cells involves PPAR-.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

15d-PGJ₂, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14643), ciglitazone (thiazolidinedione), and anti-PPAR-1,2 rabbit polyclonal Ab were purchased from Biomol (Plymouth Meeting, PA). Troglitazone (Rezulin, thiazolidinedione) was a gift from Parke-Davis (Ann Arbor, MI). PGF₂, MTT and DMSO were purchased from Sigma (St. Louis, MO). Tri-reagent was obtained from MRC (Cincinnati, OH). Annexin V-FITC conjugate was purchased from Southern Biotechnology Associates (Birmingham, AL). Propidium iodide (PI) was obtained from Roche (Indianapolis, IN).

Cell lines and culture conditions

70Z/3, ECH408.1, WEHI-231, CH31, CH12, BAL-17, and J558 are clonal B lymphoma cell lines that reflect stages of B cell ontogeny and have been described in detail previously (17, 18). Briefly, 70Z/3 is positive for cytoplasmic Ig; is negative for expression of surface Ig, MHC II, and FcR; and is a model for pre-B lymphocytes. CH31, WEHI-231, and ECH408.1 are lymphomas that express surface IgM, MHC II, FcR, and CD5; do not secrete Ig; and are models for immature B lymphocytes based on their susceptibility to anti-IgM-induced apoptosis. The BAL-17 and CH12 lymphomas resemble mature B lymphocytes in that they express surface IgM, MHC II, and FcR but do not secrete Ig, nor are they susceptible to anti-IgM-induced apoptosis. J558 is a mouse myeloma that resembles plasma cells in that it expresses PC.1, does not express surface Ig, and secretes IgA. Normal B lymphocytes and lymphoma cell lines were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT), 5 x 10^-5 M 2-ME (Eastman Kodak, Rochester, NY), 10 mM HEPES (U.S. Biochemical Corp., Cleveland, OH), 2 mM L-glutamine (Life Technologies), and 50 µg/ml gentamicin (Life Technologies). The B lymphomas were kept under the same culture conditions, except for the percentage of FBS, which was 5%. Normal and neoplastic B cells and C3H10T1/2 fibroblast-like cells were preincubated with PPAR- agonists and non-PPAR- agonists in FBS-free medium for a period of not more than 6 h (19). FBS was then added at the percentages indicated above. When normal B lymphocytes were used, the medium was also supplemented with IL-4 (500 U/ml; Genzyme, Cambridge, MA) and Escherichia coli LPS (1 µg/ml; Sigma) to drive their proliferation.

cDNA synthesis and RT-PCR for PPAR-

Total RNA was extracted with Tri-Reagent (5 x 10⁶ cells/ml) following the supplier’s protocol. RNA was solubilized in nuclease-free water by heating to 55°C for 10 min. Two micrograms of RNA was reversed transcribed using Moloney murine leukemia virus reverse transcriptase (200 U/reaction; Life Technologies, Gaithersburg, MD) and random hexamers (18). For each cDNA synthesis reaction, a parallel reaction was performed without reverse transcriptase and was used in PCR as a negative control. PCR reactions for mouse PPAR- and GAPDH were performed using Taq DNA polymerase (2.5 U; Roche). Mouse PPAR- primers sequences were 5'-CAAGACTACCCTTTACTGAA and 5'-CTACTTTGATCGCACTTTGGT. PCR samples were initially denatured at 94°C for 1 min and then run for 40 cycles (94°C for 45 s, 56°C for 45 s, 72°C for 1 min) with a final extension at 72°C for 5 min in a DNA thermal cycler (PTC-200, MJ Research, Watertown, MA). The identities of these RT-PCR products were confirmed by sequencing. No products were observed in samples lacking reverse transcriptase.

Western blot for PPAR-

Total proteins were isolated from all cell lines using Tri-Reagent and were quantified using bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Fifteen micrograms of total protein was fractionated by 10% SDS-PAGE and electrophoretically transferred to a Hybond-Extra nitrocellulose membrane (Amersham, Arlington Heights, IL). After blocking with 10% dry milk in 1x PBS/0.1% Tween 20, membranes were then incubated with anti-PPAR- (1/2000 diluted in PBST) for 2 h at room temperature. They were then briefly washed and incubated with a goat anti-rabbit-HRP conjugate (1/3000 diluted in 10% milk-PBST) for 1 h. Bands were visualized with ECL according to the manufacturer’s instructions (Amersham). The density of the resulting bends was analyzed with Kodak Digital Science software (New Heaven, CT).

Isolation of normal mouse B lymphocytes

Mouse B lymphocytes were isolated as previously described (20). In brief, normal mouse splenic B lymphocytes were isolated from 7- to 22-wk-old C57BL/6JxDBA/2J (B6D2(F₁)) male mice (The Jackson Laboratory, Bar Harbor, ME). Erythrocytes were lysed with ammonium chloride buffer, and the suspension was depleted of adherent cells by two successive rounds of adherence on Falcon tissue culture dishes (Becton Dickinson, Lincoln Park, NJ) at 37°C for 2 h. Nonadherent cells were collected by rinsing the plates with warm medium. T cells were depleted with anti-T cell cocktail consisting of Abs 30-H12 (anti-Thy 1.2), 3.155 (anti-CD8), and GK 1.5 (anti-CD4) and low toxicity baby rabbit complement (Cedarlane, Hornby, Canada) followed by incubation at 37°C. The cells were washed and counted, and viability was determined using the trypan blue exclusion method. B lymphocytes isolated in this manner are >95% surface IgM positive, are Thy-1.2 negative (as measured by indirect immunofluorescence and flow cytometry), and do not express esterase (-naphthyl acetate method) or phagocytic activity (latex bead method).

Purification of small, dense, resting B lymphocytes

B cells were separated based on the method of using a discontinuous Percoll gradient (Pharmacia, Piscataway, NJ) (21) that was modified to isolate the smallest, densest cells (20). Cells resuspended in PBS (1 x 10⁸) were applied to the gradient and centrifuged for 12 min at 3000 rpm. Cells from the lowest interface (resting B lymphocytes) were harvested. This procedure yields 30–50% of the starting population. Small, dense, resting B lymphocytes fractionated by this method are >98% surface IgM⁺ (as measured by indirect immunofluorescence and flow cytometry). The median volume of cells in the lower fraction was 105–115 fl as determined by analysis on a Coulter Channelyzer 256 (Coulter Electronics, Hialeah, FL) (22).

Viability assays

Small, resting B lymphocytes and B lymphomas were cultured in flat-bottom 96-well microtiter plates (Falcon, Becton Dickinson). Cells were incubated in triplicate at 5 x 10⁵ cell/ml with 15d-PGJ₂, PGF₂, or DMSO as a control at doses of 0.001–1000 µM for 48 h. Then a solution of 5 mg/ml of MTT in 1x PBS was added (10 µl/well). After 4 h at 37°C the plate was centrifuged, the medium was removed, and the insoluble precipitate was dissolved by adding 200 µl of DMSO to each well. The plate was read at 570 nm on the ELISA reader (Dynatech, Chantilly, VA).

Proliferation assays

An experimental set-up similar to that described above for the viability assays was used to assess proliferation. After the initial 24 h of a 48-h incubation period, cells were pulsed with 1 µCi/well of [³H]thymidine. Plates were harvested with a Micromate 196 cell harvester (Packard, Meriden CT), and incorporation of [³H]thymidine was determined with a Matrix 96 direct beta counter (Packard).

Annexin V-fluorescein

Double staining for annexin V-FITC binding and for cellular DNA using PI was performed as follows. Cells (1 x 10⁶/ml) were incubated in the presence or the absence of PPAR- agonists, irrelevant agonists, medium only, or DMSO in flat-bottom plates (Falcon) for 2–6 h, then washed twice in cold PBS. The pellet was resuspended in cold binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂, and 0.1% BSA) to a concentration of 1 x 10⁶ cells/ml. In a separate tube, 100 µl of the cell suspension was incubated for 15 min with 10 µl of annexin V-FITC. Samples were placed on ice and protected from light. Without washing, 380 µl of cold 1x binding buffer was added to each tube. Then, to each sample 100 µl of PI (0.5 mg/ml) was added before the samples were analyzed by flow cytometry. All samples were analyzed on an Beckman Coulter EPICS Elite ESP flow cytometer (Beckman Coulter, Hialeah, FL) with a 488-nm argon laser. FITC was collected through a 525-nm bandpass filter, and PI was collected through 610-nm bandpass filters. Analyses were performed with EPICS Elite software.

TUNEL assay

The TUNEL acronym stands for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragmentation sites (23). To study apoptosis by the TUNEL assay, 5.0 x 10⁵ cells/ml were incubated in the presence or the absence of PPAR- agonists and nonagonists or in medium only and DMSO as controls in flat-bottom plates (Falcon) for 6–12 h. Cells were then washed twice in 1x PBS, air-dried on glass microscope slides (VWR, Media, PA), and fixed in 4% paraformaldehyde-0.5% Tween for 10–15 min at room temperature. Next, the endogenous peroxidase activity was quenched for 15 min. To label cells, a solution containing recombinant terminal deoxynucleotidyl transferase (Life Technologies, Grand Island, NY), biotin dUTP (Roche, Indianapolis, IN), and 5x terminal deoxynucleotidyl transferase buffer (Life Technologies) was applied to experimental samples for 1 h at 37°C. The TUNEL reaction was finished with termination buffer (300 mM NaCl and 30 mM sodium citrate) followed by water rinses. Samples were blocked with 2% BSA for 10 min at room temperature and treated with streptavidin peroxidase (1/6000; Zymed, South San Francisco, CA) for 30 min followed by aminoethylcarbazole substrate (Zymed) for 15 min, coverslipped with Immuno-Mount (Shandon, Pittsburgh, PA), and photographed with an Olympus BMAX B201 microscope (New Hyde Park, NY).

Immunoprecipitation and Western blot analysis of phosphorylated mitogen-activated protein kinase (MAPK)

Nuclear protein extracts from B cells were prepared as previously described (24) and immunoprecipitated overnight using a polyclonal anti-phosphotyrosine Ab (Transduction Laboratories, Lexington, KY). Protein A-agarose was added, and samples were incubated for 1 h at 4°C. Complexes were washed with hypotonic buffer containing 0.2% Nonidet P-40, and immunoprecipitated proteins were separated by discontinuous 9% PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA). After blocking with 5% powdered milk in Tris-buffered saline containing 1% Tween, membranes were incubated with 1/1000 monoclonal anti-MAPK Ab (anti-ERK2, Upstate Biotechnology, Lake Placid, NY) overnight at room temperature and washed, and the secondary, rabbit anti-mouse IgG was added at 1/1000. The signals were visualized using an ECL detection kit.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
B-lineage cells express PPAR- mRNA and protein

15d-PGJ₂, in contrast to other PGs, is actively incorporated into the cell by unknown mechanisms and transferred into the nucleus, where it binds to PPAR- (9). To assess whether small, dense, resting B lymphocytes and B lymphoma cells express mRNA for PPAR-, RT-PCR analysis was used. The B lymphoma cells analyzed ranged from 70Z/3 (pre-B cell) to J558 (a plasmacytoma). As shown in Fig. 1, normal splenic B cells and all B lymphoma cells tested yielded a 250-bp product characteristic of PPAR- mRNA. C3H10T1/2 cells, a fibroblast-like line, was used as a positive control for PPAR- mRNA (9). Normal B cells and the same panel of B lymphoma cells as that used for RT-PCR analysis were lysed and analyzed for PPAR- protein by Western blot using a polyclonal Ab. PPAR- appeared as a single band with a characteristic molecular mass of 67 kDa on all the B-lineage cells (Fig. 2A). While each cell type expressed mRNA for PPAR-, the relative amount of PPAR- protein did not vary significantly in B lymphoma cells at different stages of differentiation, as shown by the Western blot (Fig. 2B). Normal splenic B cells, however, did express a relatively larger amount of PPAR- protein.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. B-lineage cells express mRNA for PPAR-. Two micrograms of RNA was reversed transcribed using Moloney murine leukemia virus reverse transcriptase and random hexamers (see Materials and Methods). PCR reactions for mouse PPAR- were completed using Taq DNA polymerase and mouse PPAR- primers. The expected product was 250 bp. The identities of these products were confirmed by sequencing. No products were observed in samples without reverse transcriptase.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. A, B-lineage cells express PPAR- protein. Fifteen micrograms of total cellular protein was fractionated by 10% SDS-PAGE and electrophoretically transferred to a Hybond-Extra nitrocellulose membrane (see Materials and Methods). B lymphomas and controls were all cultivated and lysed in the same experiment. Membranes were incubated with anti-PPAR- polyclonal Ab, washed, and incubated with a goat anti-rabbit-HRP conjugate. Bands were visualized with ECL. B, PPAR- protein levels did not vary among the B lymphoma cell lines. The relative intensities of three separate PPAR- Western blots using the same assortment of B cell lymphomas and control cell lines were analyzed and plotted.

15d-PGJ₂ is the only physiological ligand, known to date to bind PPAR- (9, 25). [³H]thymidine incorporation assays were used to determine whether 15d-PGJ₂ had an effect on proliferation of B-lineage cells. 15d-PGJ₂ was used from 0.001–10 µM for 48 h, and PGF₂ was included as a negative control prostanoid. Murine B lymphoma cell lines representative of different stages of B cell ontogeny were used as well as small, dense, resting mouse B cells. Fig. 3 reveals the antiproliferative effect that 15d-PGJ₂ had on B cell lines at 1 µM. 15d-PGJ₂ inhibited [³H]thymidine incorporation in the different B cell lines regardless of their stage of development. Normal B lymphocytes and lymphoma cells were homogeneous in their sensitivity to the antiproliferative effect caused by 15d-PGJ₂ at 1 µM. The incorporation of [³H]thymidine was not inhibited by the presence of PGF₂. C3H10T1/2 were not inhibited in their [³H]thymidine incorporation by either 15d-PGJ₂ or PGF₂ at 1 µM. To determine whether the antiproliferative effect that 15d-PGJ₂ has on B-lineage cells is cytotoxic, MTT assays were performed. The MTT assay evaluates cell viability via cleavage of MTT into a dark blue crystalline compound by mitochondrial succinic dehydrogenases active only in living cells. Fig. 4 depicts the effects of 15d-PGJ₂ on the viability of the different B cell lines. Once again 15d-PGJ₂ had a uniformly negative effect on B-lineage cells regardless of the stage of B cell ontogeny. Therefore, 15d-PGJ₂ is a negative regulator of B-lineage cells.

View larger version (63K): [in this window] [in a new window]   FIGURE 3. 15d-PGJ2 induces growth inhibition of B-lineage cells. [3H]Thymidine incorporation assay of B-lineage cells and C3H10T1/2 fibroblast-like cells treated with 15d-PGJ2 or PGF2 at 1 µM for 48 h. After the initial 24-h incubation period, cells were pulsed with 1 µCi/well of [3H]thymidine. Plates were harvested with a Micromate 196 cell harvester, and incorporation of [3H]thymidine was determined with a Matrix 96 direct beta counter (see Materials and Methods). These data were graphed as a percentage of the vehicle (DMSO) control response.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. 15d-PGJ2 kills B-lineage cells. A viability assay was performed on B-lineage cells and C3H10T1/2 cells treated with 15d-PGJ2 or PGF2 at 1 µM for 48 h. B-lineage cells are homogeneous in their sensitivity to 15d-PGJ2. In contrast, C3H10T1/2 are not killed by 15d-PGJ2. After 48 h of incubation a solution of 5 mg/ml of MTT was added. Plates were kept at 37°C for 4 h, then centrifuged, and the precipitate was dissolved by adding 200 µl of DMSO to each well. Plates were read at 570 nm on an ELISA reader (see Materials and Methods). These data were graphed as a percentage of the DMSO control response.

The mechanism of 15d-PGJ₂ cytotoxicity on B-lineage cells involves PPAR-

To investigate whether 15d-PGJ₂ involves PPAR- as the mechanism for the cytotoxic effect, we used additional known PPAR activators. Troglitazone and ciglitazone are both antihyperglycemic agents that are PPAR--selective activators (9, 26). WY14,643 is a fibrate hypolipidemic drug that activates PPAR selectively (27), and eicosatetraynoic acid is a synthetic arachidonic acid analogue that binds PPAR- selectively (28). PGF₂ is used as a negative control prostanoid, because it does not bind to PPARs (9, 25). [³H]thymidine incorporation assays were performed, and representative data using CH31 cells are depicted in Fig. 5. 15d-PGJ₂ inhibited the incorporation of [³H]thymidine in a dose-dependent manner, with total inhibition at 1 µM. The PPAR--selective agonists ciglitazone and troglitazone mimicked the effect of 15d-PGJ₂ on the inhibition of [³H]thymidine incorporation. Troglitazone and ciglitazone were 10-fold less potent than 15d-PGJ₂ in their antiproliferative effect. This is similar to the increased ability of 15d-PGJ₂ compared with ciglitazone and troglitazone to bind PPAR- (11, 26). Non-PPAR- agonists such as PGF₂, WY14,643, and eicosatetraynoic acid (data not shown) failed to inhibit the proliferation of CH31 cells. These data indicate that the mechanism by which 15d-PGJ₂ interacts with B-lineage cells involves in part PPAR-. These experiments were repeated with other B lymphomas, such as 70Z/3, CH12, and ECH408.1, with similar results.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. PPAR- agonists (ciglitazone and troglitazone) mimic the effect that 15d-PGJ2 has on CH31 cells. Non-PPAR- agonists (PGF2 and WY14,643) have no antiproliferative effect on CH31 cells. A [3H]thymidine incorporation assay was performed on CH31 cells treated with PPAR- agonists and negative control agonists for 48 h. After the initial 24-h incubation period, cells were pulsed with 1 µCi/well of [3H]thymidine. Plates were harvested with a Micromate 196-cell harvester, and incorporation of [3H]thymidine was determined with a Matrix 96 direct beta counter (see Materials and Methods). These data were graphed as a percentage of the DMSO control response.

Death by apoptosis is characterized by key morphological features. These include changes in the plasma membrane, such as loss of membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA by an endogenous endonuclease. In the final stages the dying cells become fragmented into apoptotic bodies, which are rapidly eliminated by phagocytic cells without eliciting significant inflammatory damage to surrounding cells. To evaluate whether 15d-PGJ₂ kills B cells by inducing cell death in an apoptotic fashion we first examined the translocation of phosphatidylserine from the inner leaflet of the plasma membrane to the outer leaflet by annexin V-FITC labeling. Annexin V is a Ca²⁺-dependent, phospholipid-binding protein with high affinity for phosphatidylserine (29). Annexin V-FITC in concert with flow cytometry is a sensitive method to detect cells that are undergoing early phases of apoptosis (Fig. 6). WEHI-231 cells were incubated in the presence of medium or the PPAR- agonists, 15d-PGJ₂ (1 µM) and troglitazone (10 µM; Fig. 6, A, B, and C, respectively), and the non-PPAR- agonists, WY14,643 and PGF₂ (data not shown). Samples were labeled simultaneously with annexin V-FITC and PI. The viable cells depicted in Fig. 6A (box 3) are double negative (FITC^-/PI^-). Cells at an early stage of apoptosis are positive for FITC but negative for PI (FITC⁺/PI^-; box 4). WEHI-231 cells treated with 15d-PGJ₂ or troglitazone showed a population of cells that was apoptotic. Also shown (box 2) were cells that were double positive (FITC⁺/PI⁺). These cells were at later stages of apoptosis and showed evidence of necrosis, as they were permeable to PI. According to these data both PPAR- agonists induced apoptosis in WEHI-231 cells. Fig. 7 shows similar results for CH31 cells that were incubated in the presence of medium or the PPAR- agonists, 15d-PGJ₂ (1 µM), ciglitazone (10 µM), and troglitazone (10 µM; Fig. 7, A, B, C, and D, respectively), and the non-PPAR- agonists, WY14,643 and PGF₂ (data not shown). CH31 cells treated with 15d-PGJ₂, ciglitazone, or troglitazone showed a population of cells that was apoptotic. According to these data PPAR- agonists induced apoptosis in CH31 cells.

View larger version (59K): [in this window] [in a new window]   FIGURE 6. PPAR- agonists induce apoptosis in WEHI-231 B lymphoma cells. WEHI-231 cells were incubated with PPAR- agonists or with negative control agonists (e.g., PPAR- agonist) for 6–12 h. Fresh untreated cells (A and D) or those incubated with 15d-PGJ2 (1 µM; B and E) or troglitazone (10 µM; C and F) are shown. Apoptosis hallmarks were detected by annexin V-FITC (B and C) and were confirmed by TUNEL assay (E and F; see Materials and Methods). No annexin V-FITC labeling or BdUTP incorporation was detected on samples incubated with the PPAR- agonist WY14,643 and/or with PGF2.

View larger version (67K): [in this window] [in a new window]   FIGURE 7. PPAR- agonists induce apoptosis in CH31 B lymphoma cells. CH31 cells were incubated with PPAR- agonists or with negative control agonists (e.g., PPAR- agonist) for 6–12 h. Fresh untreated cells (A and E) or those incubated with 15d-PGJ2 (1 µM; B and F), ciglitazone (10 µM; C and G), or troglitazone (10 µM; D and H) are shown. Apoptosis hallmarks were detected by annexin V-FITC (B–D) and were confirmed by TUNEL assay (F–H; see Materials and Methods). No annexin V-FITC labeling or BdUTP incorporation was detected on samples incubated with the PPAR- agonist WY14,643 and/or with PGF2.

2

PGF₂ abrogates the induction of apoptosis induced by PPAR- agonists on B lymphoma cells

PGF₂ was recently proposed to modulate the adipogenic effect of 15d-PGJ₂ (15, 30). The mechanism by which PGF₂ interfered with PPAR-/PPAR- agonist interaction involved MAPK (30). To further evaluate the mechanisms involved in the interaction between 15d-PGJ₂ and PPAR- in B lymphocytes, we preincubated B lymphoma cells (CH31, ECH.408.1, and J558) with PGF₂ (0.1 µM) for 1 h before treatment with 15d-PGJ₂ (1 µM). [³H]thymidine incorporation assays were used to determine whether PGF₂ modulated the activity of 15d-PGJ₂ (Fig. 8A). PGF₂ strongly blocked the inhibitory action of 15d-PGJ₂ on these B lymphoma cells, allowing cell proliferation to proceed normally, as detected by incorporation of [³H]thymidine. This was interesting, as PGF₂ was not known to have any effect on lymphocytes. We next examined the effect that these prostanoids had on MAPK activation of B-lineage cells. Fig. 8B shows the dramatic changes in the levels of MAPK activity reflecting the opposing effects of PGF₂ and 15d-PGJ₂. Treatment with 15d-PGJ₂ (1 µM) induced a massive increase in MAPK (Fig. 8B). This was a rather surprising finding, as 15d-PGJ₂ is not known to activate MAPK in nonhemopoietic cells. PGF₂ (0.1 µM), however, is known to activate MAPK (30) and does so in B-lineage cells (Fig. 8B). Pretreatment with PGF₂ followed by 15d-PGJ₂ (1 µM) dramatically reduced the ability of 15d-PGJ₂ to induce MAPK activation (Fig. 8B). Levels of MAPK activation of untreated cells were negligible.

View larger version (32K): [in this window] [in a new window]   FIGURE 8. PGF2 abrogates the deleterious effects of PPAR- agonist on B lymphoma cells. A, The B lymphoma cells CH31, ECH.408.1, and J558 were preincubated with PGF2 (0.1 µM) for 1 h before treatment with 15d-PGJ2 (1 µM) for 24 h. Cells were also treated with only 15d-PGJ2 or PGF2. B lymphoma cells were pulsed with 1 µCi/well of [3H]thymidine (see Materials and Methods). B, After treatment with medium, 15d-PGJ2, or PGF2 alone or with PGF2 plus 15d-PGJ2, nuclei from J558 B cells were harvested. Immunoprecipitation and Western blot of phosphorylated MAPK were performed (see Materials and Methods). 15d-PGJ2 dramatically induced MAPK activation. PGF2 induced a more modest activation of MAPK. Pretreatment with PGF2 caused a substantial attenuation of the 15d-PGJ2-induced MAPK activation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

PGF₂ signals through a cell surface receptor and does not activate PPAR- (9, 25). PGF₂ has an inhibitory effect on adipocyte differentiation, which involves MAPK (15, 30). In our investigation PGF₂ strongly modulated the deleterious effect that 15d-PGJ₂ had on B-lineage cells. Therefore, 15d-PGJ₂ is not simply a toxic molecule, because PGF₂ attenuates its proapoptotic affects. We were surprised to see that 15d-PGJ₂ induced a massive MAPK activation. PGF₂ pretreatment caused a substantial attenuation of the 15d-PGJ₂-induced MAPK. These data support the concept that the mechanism by which 15d-PGJ₂ interacts with B cells involves PPAR-. However, we cannot disregard the possibility that 15d-PGJ₂ may have other effects on B-lineage cells that may be PPAR- independent, as reported by others studying microglial inducible NO synthase (36) and neutrophils (37). We speculate that the mechanism by which PPAR- agonists interact with B lymphocytes and induce apoptosis involves the activation of MAPK. Additional research will need to be performed to dissert the precise molecular pathways involved in PPAR- agonist-induced signal transduction. Finally, we suggest a scenario in which alteration of the 15d-PGJ₂/PGF₂ balance could affect the development of B cell lymphomas. Environments rich in PGF₂ would blunt the ability of 15d-PGJ₂ to kill B-lineage cells. Alternatively, the use of 15d-PGJ₂ or its analogues in the appropriate prostanoid milieu may be useful as a therapy for B-lineage malignancies.

	Acknowledgments

 
We thank Parke-Davis for the generous gift of troglitazone.

	Footnotes

 
¹ This work was supported by U.S. Public Health Service Grants 5-T32DE07061–21, DE11390, CA11198, HL56002, ES01247, EY08976, and EY11708 and a University of Rochester Cancer Center Discovery Fund Grant.

² Address correspondence and reprint requests to Dr. Richard P. Phipps, Box 704, Cancer Center, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642.

³ Abbreviations used in this paper: 15d-PGJ₂, 15-deoxy-^12,14-PGJ₂; PPAR-, peroxisome proliferator-activated receptor ; MTT, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide; WY-14643, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid; PI, propidium iodide; MAPK, mitogen-activated protein kinase; BdUTP, bromo-dUTP.

Received for publication March 7, 2000. Accepted for publication September 20, 2000.

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